Low-Dose Ethanol Preconditioning Protects Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Injury By Activating Large Conductance, Ca2+-Activated K+ Channels In Vitro.
ABSTRACT: Increasing evidence suggests that low to moderate ethanol ingestion protects against the deleterious effects of subsequent ischemia/reperfusion; however, the underlying mechanism has not been elucidated. In the present study, we showed that expression of the neuronal large-conductance, Ca2+-activated K+ channel (BKCa) ?-subunit was upregulated in cultured neurons exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) compared with controls. Preconditioning with low-dose ethanol (10 mmol/L) increased cell survival rate in neurons subjected to OGD/R, attenuated the OGD/R-induced elevation of cytosolic Ca2+ levels, and reduced the number of apoptotic neurons. Western blots revealed that ethanol preconditioning upregulated expression of the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic protein Bax. The protective effect of ethanol preconditioning was antagonized by a BKCa channel inhibitor, paxilline. Inside-out patches in primary neurons also demonstrated the direct activation of the BKCa channel by 10 mmol/L ethanol. The above results indicated that low-dose ethanol preconditioning exerts its neuroprotective effects by attenuating the elevation of cytosolic Ca2+ and preventing neuronal apoptosis, and this is mediated by BKCa channel activation.
Project description:Electrical activity in neurons and other excitable cells is a result of complex interactions between the system of ion channels, involving both global coupling (e.g., via voltage or bulk cytosolic Ca2+ concentration) of the channels, and local coupling in ion channel complexes (e.g., via local Ca2+ concentration surrounding Ca2+ channels (CaVs), the so-called Ca2+ nanodomains). We recently devised a model of large-conductance BKCa potassium currents, and hence BKCa-CaV complexes controlled locally by CaVs via Ca2+ nanodomains. We showed how different CaV types and BKCa-CaV stoichiometries affect whole-cell electrical behavior. Ca2+ nanodomains are also important for triggering exocytosis of hormone-containing granules, and in this regard, we implemented a strategy to characterize the local interactions between granules and CaVs. In this study, we coupled electrical and exocytosis models respecting the local effects via Ca2+ nanodomains. By simulating scenarios with BKCa-CaV complexes with different stoichiometries in pituitary cells, we achieved two main electrophysiological responses (continuous spiking or bursting) and investigated their effects on the downstream exocytosis process. By varying the number and distance of CaVs coupled with the granules, we found that bursting promotes exocytosis with faster rates than spiking. However, by normalizing to Ca2+ influx, we found that bursting is only slightly more efficient than spiking when CaVs are far away from granules, whereas no difference in efficiency between bursting and spiking is observed with close granule-CaV coupling.
Project description:Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa) of smooth muscle cells (VSMC) from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming ?-subunit. BKCa variants in the intracellular C terminus of the ?-subunit, and their relative expression to total ?-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two ?-subunit variants, ZERO and STREX, to be identical in cremaster and cerebral arteries. Levels of STREX mRNA expression were, however, significantly higher in cremaster VSMCs (28.9±4.2% of total ?-BKCa) compared with cerebral vessels (16.5±0.9%). Further, a low level of BKCa SS4 ?-subunit variant was seen in cerebral arteries, while undetectable in cremaster arteries. Protein biotinylation assays, in expression systems and arterial preparations, were used to determine whether differences in splice variant mRNA expression affect surface membrane/cytosolic location of the channel. In AD-293 and CHO-K1 cells, rat STREX was more likely to be located at the plasma membrane compared to ZERO, although the great majority of channel protein was in the membrane in both cases. Co-expression of ?1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of ?-BKCa subunits, whereas in the absence of ?-BKCa, a significant proportion of ?1-subunit remained cytosolic. Biotinylation assays of cremaster and cerebral arteries showed that differences in STREX/ZERO expression do not alter membrane/cytosolic distribution of the channel under basal conditions. These data, however, revealed that the amount of ?-BKCa in cerebral arteries is approximately 20X higher than in cremaster vessels. Thus, the data support the major functional differences in BKCa activity in cremaster, as compared to cerebral VSMCs, being related to total ?-BKCa expression, regardless of differences in splice variant expression.
Project description:Tolerance to otherwise lethal cerebral ischemia in vivo or to oxygen-glucose deprivation (OGD) in vitro can be induced by prior transient exposure to N-methyl-D-aspartic acid (NMDA): preconditioning in this manner activates extrasynaptic and synaptic NMDA receptors and can require bringing neurons to the "brink of death." We considered if this stressful requirement could be minimized by the stimulation of primarily synaptic NMDA receptors. Subjecting cultured cortical neurons to prolonged elevations in electrical activity induced tolerance to OGD. Specifically, exposing cultures to a K(+)-channel blocker, 4-aminopyridine (20-2500 microm), and a GABA(A) receptor antagonist, bicuculline (50 microm) (4-AP/bic), for 1-2 days resulted in potent tolerance to normally lethal OGD applied up to 3 days later. Preconditioning induced phosphorylation of ERK1/2 and CREB which, along with Ca(2+) spiking and OGD tolerance, was eliminated by tetrodotoxin. Antagonists of NMDA receptors or L-type voltage-gated Ca(2+) channels (L-VGCCs) applied during preconditioning decreased Ca(2+) spiking, phosphorylation of ERK1/2 and CREB, and OGD tolerance more effectively when combined, particularly at the lowest 4-AP concentration. Inhibiting ERK1/2 or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) also reduced Ca(2+) spiking and OGD tolerance. Preconditioning resulted in altered neuronal excitability for up to 3 days following 4-AP/bic washout, based on field potential recordings obtained from neurons cultured on 64-channel multielectrode arrays. Taken together, the data are consistent with action potential-driven co-activation of primarily synaptic NMDA receptors and L-VGCCs, resulting in parallel phosphorylation of ERK1/2 and CREB and involvement of CaMKs, culminating in a potent, prolonged but reversible, OGD-tolerant phenotype.
Project description:We investigated whether thrombin preconditioning of human Wharton's jelly-derived mesenchymal stem cells (MSCs) improves paracrine potency and thus the therapeutic efficacy of naïve MSCs against severe hypoxic ischemic encephalopathy (HIE). Thrombin preconditioning significantly enhances the neuroprotective anti-oxidative, anti-apoptotic, and anti-cytotoxic effects of naïve MSCs against oxygen-glucose deprivation (OGD) of cortical neurons in vitro. Severe HIE was induced in vivo using unilateral carotid artery ligation and hypoxia for 2 h and confirmed using brain magnetic resonance imaging (MRI) involving >40% of ipsilateral hemisphere at postnatal day (P) 7 in newborn rats. Delayed intraventricular transplantation of 1 × 105 thrombin preconditioned but not naïve MSCs at 24 h after hypothermia significantly enhanced observed anti-inflammatory, anti-astroglial, and anti-apoptotic effects and the ensuing brain infarction; behavioral tests, such as cylinder rearing and negative geotaxis tests, were conducted at P42. In summary, thrombin preconditioning of human Wharton's jelly-derived MSCs significantly boosted the neuroprotective effects of naïve MSCs against OGD in vitro by enhancing their anti-oxidative, anti-apoptotic, and anti-cytotoxic effects, and significantly attenuated the severe HIE-induced brain infarction and improved behavioral function tests in vivo by maximizing their paracrine anti-inflammatory, anti-astroglial, and anti-apoptotic effects.
Project description:Excitotoxic mechanisms contribute to the degeneration of hippocampal pyramidal neurons after recurrent seizures and brain ischemia. However, susceptibility differs, with CA1 neurons degenerating preferentially after global ischemia and CA3 neurons after limbic seizures. Whereas most studies address contributions of excitotoxic Ca2+ entry, it is apparent that Zn2+ also contributes, reflecting accumulation in neurons either after synaptic release and entry through postsynaptic channels or upon mobilization from intracellular Zn2+-binding proteins such as metallothionein-III (MT-III). Using mouse hippocampal slices to study acute oxygen glucose deprivation (OGD)-triggered neurodegeneration, we found evidence for early contributions of excitotoxic Ca2+ and Zn2+ accumulation in both CA1 and CA3, as indicated by the ability of Zn2+ chelators or Ca2+ entry blockers to delay pyramidal neuronal death in both regions. However, using knock-out animals (of MT-III and vesicular Zn2+ transporter, ZnT3) and channel blockers revealed substantial differences in relevant Zn2+ sources, with critical contributions of presynaptic release and its permeation through Ca2+- (and Zn2+)-permeable AMPA channels in CA3 and Zn2+ mobilization from MT-III predominating in CA1. To assess the consequences of the intracellular Zn2+ accumulation, we used OGD exposures slightly shorter than those causing acute neuronal death; under these conditions, cytosolic Zn2+ rises persisted for 10-30 min after OGD, followed by recovery over ?40-60 min. Furthermore, the recovery appeared to be accompanied by mitochondrial Zn2+ accumulation (via the mitochondrial Ca2+ uniporter MCU) in CA1 but not in CA3 neurons and was markedly diminished in MT-III knock-outs, suggesting that it depended upon Zn2+ mobilization from this protein.The basis for the differential vulnerabilities of CA1 versus CA3 pyramidal neurons is unclear. The present study of events during and after acute oxygen glucose deprivation highlights a possible important difference, with rapid synaptic entry of Ca2+ and Zn2+ contributing more in CA3, but with delayed and long-lasting accumulation of Zn2+ within mitochondria occurring in CA1 but not CA3 pyramidal neurons. These data may be consistent with observations of prominent mitochondrial dysfunction as a critical early event in the delayed degeneration of CA1 neurons after ischemia and support a hypothesis that mitochondrial Zn2+ accumulation in the early reperfusion period may be a critical and targetable upstream event in the injury cascade.
Project description:KEY POINTS:Association of plasma membrane BKCa channels with BK-? subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa ) by BK-? subunits is not established. MitoBKCa -? and the regulatory BK-?1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK-?1 (left-shifted voltage dependence of activation, V1/2 = -55 mV, 12 µm matrix Ca2+ ). In BK-?1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+ ) Co-expression of BKCa with the BK-?1 subunit in HeLa cells doubled the density of BKCa in mitochondria. The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK-?1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. ABSTRACT:Association of the plasma membrane BKCa channel with auxiliary BK-?1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa ) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK-?1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBKCa displays a high open probability (Po ) and voltage half-activation (V1/2 = -55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK-?1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa -? and its BK-?1 subunit. Mitochondria from the BK-?1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V1/2 of activation (+47 mV in 12 µm matrix Ca2+ ). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK-?1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK-?1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-?1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK-?1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.
Project description:The anterior cingulate cortex (ACC) serves as a critical hub for the anxiety and pain perception. The large-conductance Ca2+-activated potassium channels, or BKCa channels, are ubiquitously expressed throughout the central nervous system including the cingulate cortex. However, what changes of cortical BKCa channels undergo in the ACC remains unknown in pain-related anxiety. In the present study, a significant upregulation of synaptic and non-synaptic BKCa channel accessory ?4 subunits in the ACC was accompanied with pain-associated anxiety-like behaviors in the chronic compression of multiple dorsal root ganglia (mCCD) of the rat. NS1619, an opener of BKCa channels, significantly rescued the alteration of fAHP and AP duration of ACC pyramidal neurons in mCCD rats. The mRNA expression of BKCa ?4 subunits was extremely upregulated in the ACC after mCCD with the increased amount of both synaptic and non-synaptic BKCa ?4 subunit protein. Meanwhile, NS1619 reversed the enhanced AMPA receptor-mediated spontaneous excitatory postsynaptic current (sEPSC) frequency and the attenuated PPR of ACC neurons in mCCD rats. Local activation of BKCa channels in the ACC reversed mechanical allodynia and anxiety-like behaviors. These results suggest that the upregulation of postsynaptic and presynaptic BKCa ?4 subunit may contribute to neuronal hyperexcitability and the enhanced synaptic transmission in the ACC in neuropathic pain state, and then may result in anxiety-like behavior induced by neuropathic pain.
Project description:Hypoxic-ischemic brain injury in premature infants results in cerebral white matter lesions with prominent oligodendroglial injury and loss, a disorder termed periventricular leukomalacia (PVL). We have previously shown that glutamate receptors mediate hypoxic-ischemic injury to oligodendroglial precursor cells (OPCs) in a model of PVL in the developing rodent brain. We used primary OPC cultures to examine the mechanism of cellular toxicity induced by oxygen-glucose deprivation (OGD) to simulate brain ischemia. OPCs were more sensitive to OGD-induced toxicity than mature oligodendrocytes, and OPC toxicity was attenuated by nonselective [2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX), 6-cyano-7-nitroquinoxaline-2,3-dione], alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring (GYKI 52466), kainate-preferring (gamma-d-glutamylaminomethanesulfonic acid), or Ca2+-permeable AMPA/kainate receptor antagonists (joro spider toxin, JSTx) administered either during or after OGD. Furthermore, NBQX or JSTx blocked OGD-induced Ca2+ influx. Relevant to recurrent hypoxic-ischemic insults in developing white matter, we examined the effects of sublethal OGD preconditioning. A prior exposure of OPCs to sublethal OGD resulted in enhanced vulnerability to subsequent excitotoxic or OGD-induced injury associated with an increased Ca2+ influx. AMPA/kainate receptor blockade with NBQX or JSTx either during or after sublethal OGD prevented its priming effect. Furthermore, OGD preconditioning resulted in a down-regulation of the AMPA receptor subunit GluR2 on cell surface that increased Ca2+ permeability of the receptors. Overall, these data suggest that aberrantly enhanced activation of Ca2+-permeable AMPA/kainate receptors may be a major mechanism in acute and repeated hypoxic-ischemic injury to OPCs in disorders of developing cerebral white matter, such as PVL.
Project description:In this study, cerebral cortical astrocytes and neurons of rats were co-cultured, the astrocytes of the co-cultured cell model pretreated by ischemic preconditioning (45 min of OGD) were subjected to lncRNA high-throughput sequencing (RNA-seq). Overall design: Cortical astrocytes lncRNA profiles of ischemic preconditioning (45 min of OGD) and control group
Project description:Bladder small DRG neurons, which are putative nociceptors pivotal to urinary bladder function, express more than a dozen different ionic membrane mechanisms: ion channels, pumps and exchangers. Small-conductance Ca2+-activated K+ (SKCa) channels which were earlier thought to be gated solely by intracellular Ca2+ concentration ([Ca]i) have recently been shown to exhibit inward rectification with respect to membrane potential. The effect of SKCa inward rectification on the excitability of these neurons is unknown. Furthermore, studies on the role of KCa channels in repetitive firing and their contributions to different types of afterhyperpolarization (AHP) in these neurons are lacking. In order to study these phenomena, we first constructed and validated a biophysically detailed single compartment model of bladder small DRG neuron soma constrained by physiological data. The model includes twenty-two major known membrane mechanisms along with intracellular Ca2+ dynamics comprising Ca2+ diffusion, cytoplasmic buffering, and endoplasmic reticulum (ER) and mitochondrial mechanisms. Using modelling studies, we show that inward rectification of SKCa is an important parameter regulating neuronal repetitive firing and that its absence reduces action potential (AP) firing frequency. We also show that SKCa is more potent in reducing AP spiking than the large-conductance KCa channel (BKCa) in these neurons. Moreover, BKCa was found to contribute to the fast AHP (fAHP) and SKCa to the medium-duration (mAHP) and slow AHP (sAHP). We also report that the slow inactivating A-type K+ channel (slow KA) current in these neurons is composed of 2 components: an initial fast inactivating (time constant ? 25-100 ms) and a slow inactivating (time constant ? 200-800 ms) current. We discuss the implications of our findings, and how our detailed model can help further our understanding of the role of C-fibre afferents in the physiology of urinary bladder as well as in certain disorders.